Thomas A. Marino

Hemodynamic versus adrenergic control of cat right ventricular hypertrophy.

The purpose of this study was to determine whether cardiac hypertrophy in response to hemodynamic overloading is a primary result of the increased load or is instead a secondary result of such other factors as concurrent sympathetic activation. To make this distinction, four experiments were done; the major experimental result, cardiac hypertrophy, was assessed in terms of ventricular mass and cardiocyte cross-sectional area. In the first experiment, the cat right ventricle was loaded differentially by pressure overloading the ventricle, while unloading a constituent papillary muscle; this model was used to ask whether any endogenous or exogenous substance caused uniform hypertrophy, or whether locally appropriate load responses caused ventricular hypertrophy with papillary muscle atrophy. The latter result obtained, both when each aspect of differential loading was simultaneous and when a previously hypertrophied papillary muscle was unloaded in a pressure overloaded right ventricle. In the second experiment, epicardial denervation and then pressure overloading was used to assess the role of local neurogenic catecholamines in the genesis of hypertrophy. The degree of hypertrophy caused by these procedures was the same as that caused by pressure overloading alone. In the third and fourth experiments, beta-adrenoceptor or alpha-adrenoceptor blockade was produced before and maintained during pressure overloading. The hypertrophic response did not differ in either case from that caused by pressure overloading without adrenoceptor blockade. These experiments demonstrate the following: first, cardiac hypertrophy is a local response to increased load, so that any factor serving as a mediator of this response must be either locally generated or selectively active only in those cardiocytes in which stress and/or strain are increased; second, catecholamines are not that mediator, in that adrenergic activation is neither necessary for nor importantly modifies the cardiac hypertrophic response to an increased hemodynamic load.

Load regulation of the properties of adult feline cardiocytes. The role of substrate adhesion.

We have recently described rapid and reversible changes in cardiac structure, function, and composition in response to surgical load alteration in vivo. In the present study, weused a simple, well- defined in vitro experimental model system, consisting of terminally differentiated quiescent adult cat ventricular cardiocytes maintained in serum-free culture medium, to assess more definitively the role of loading conditions in regulating these same biological properties of heart muscle. Cardiocytes considered to be externally loaded were adherent throughout their length to a protein substrate, such that the tendency for the ends of the cells to retract was prevented. Cardiocytes considered to be unloaded were not adherent to a substrate and, thus, were free to assume a spherical shape. Cardiocyte structure and surface area were assessed, in initially identified cells, both by serial light microscopy and by terminal electron microscopy. Cardiocyte function was assessed in terms of the ability to exclude trypan blue, to remain quiescent with relaxed sarcomeres containing I-bands, and to shorten in response to electrical stimulation. Cardiocyte composition was first assessed by quantitative gel electrophoresis of proteins and then by microfluorimetric measurement of ribonucleic acid, protein, and deoxyribonucleic acid. In addition, cardiocyte incorporation of [3H]thymidine into deoxyribonucleic acid and [3H]uridine into ribonucleic acid were measured. Loading via substrate adhesion was found to be very effective in terms of each of these measurements in retaining the differentiated features of adult cardiocytes for up to 2 weeks in culture; unattached and thus unloaded cardiocytes quickly dedifferentiated. Conditions thought to stimulate cardiac growth, including catecholamine stimulation, were found to be ineffective. These experiments demonstrate that external load has a primary role in the maintenance of the basic differentiated properties of adult mammalian cardiocytes.

Atrophy reversal and cardiocyte redifferentiation in reloaded cat myocardium.

We have recently described rapid cardiac atrophy in response to decreased load. The present study was designed to determine whether this atrophy is solely a degenerative response of damaged myocardium or is, instead, an adaptive response of viable myocardium. A discrete portion of cat myocardium was unloaded by severing the chordae tendinae of a single right ventricular papillary muscle. One week later, the muscle was reloaded by attachment of its apex to the ventricular free wall. This allowed the load to be removed and restored without altering the blood supply, innervation, or frequency of contraction of the tissue. In myocardium unloaded for 1 week, the cardiocyte cross-sectional area and the volume densities of mitochondria and myofibrils decreased significantly. Large areas of cytoplasm were devoid of organelles, and the few remaining myofilaments were oriented in a variety of directions rather than longitudinally within the cell. Upon reloading for 1 week, the cardiocyte cross-sectional area, volume density of mitochondria, and ultrastructural organization all returned to normal. The volume density of the myofibrils increased toward control, and they reoriented with respect to the long axis of the cardiocyte. The contractile function of the papillary muscles, which was depressed as early as 1 day after unloading and almost absent at times later than 3 days after unloading, returned to normal after 2 weeks of reloading. This study demonstrates that adult mammalian myocardium responds to unloading with a marked loss of cellular differentiation, organization, and function which is fully reversible with reloading. This plasticity in response to load may well be the basic mechanism responsible for the development and maintenance of normal cardiac structure and function.

Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells.

During early development, rat cardiac muscle cells actively proliferate. Shortly after birth, division of cardiac muscle cells ceases, whereas DNA synthesis continues for approximately 2 weeks at a progressively diminishing rate. Little DNA synthesis or cell division occurs in adult cardiocytes. Thus, developing cardiac muscle cells are an ideal system in which to examine the expression of cell cycle-regulated genes during development. We chose to examine proliferating cell nuclear antigen (PCNA), a gene expressed at the G1/S phase boundary of the cell cycle. Northern blots of RNA from cardiac muscle cells from 18-day-old rat fetuses and from day 0, 5, and 14 neonatal as well as adult rat hearts revealed that the PCNA mRNA was found in cardiac muscle cells from all ages. However, because it was possible that this was a result of fibroblast PCNA gene expression, we used reverse transcription followed by polymerase chain reaction to see if it was possible to detect the message for PCNA in cardiac muscle cells from all ages. Because of the great sensitivity of this technique, RNA was recovered from 25 isolated adult cardiac muscle cells. Polymerase chain reaction amplification products for PCNA produced from the RNA isolated from these cells conclusively demonstrated that mRNA for this gene, which normally is associated with proliferating cells, is expressed in adult cardiac muscle cells that no longer divide. Furthermore, Western blot analysis demonstrated that the PCNA protein was found only in embryonic and neonatal cells and not in adult rat cardiac muscle cells. Therefore, it might be inferred from these data that PCNA might be regulated at the posttranscriptional level in adult cardiac muscle cells.

Biochemical and structural correlates in unloaded and reloaded cat myocardium

Cardiocytes of unloaded myocardium rapidly lose structural and functional integrity through a combined loss of myofibrils and contractile activity; both changes are reversible with load restoration. The present study correlates the biochemical composition of unloaded and reloaded myocardium with these alterations in structure and function. Cardiac muscle was unloaded by transecting the chordae tendineae of a cat right ventricular papillary muscle and was reloaded by suturing these same chordae tendineae to the ventricular wall at the base of the valve; an adjacent intact muscle served as the control. Muscles unloaded for 1 to 14 days were assayed for DNA, protein, total creatine and hydroxyproline content. The ratios of wet weight/DNA and creatine/DNA decreased by 30 and 22% respectively, in parallel with a 38% reduction in cardiocyte cross-sectional area. Protein/unit wet weight was decreased by 50% after 14 days of unloading, so that both protein/DNA and protein/creatine were markedly reduced. Reloading of the muscle restored cardiocyte size, protein per unit wet weight and protein/DNA to normal. Parallel reductions in both contractile filaments and contractile proteins after unloading and parallel increases in each following load restoration were demonstrated by morphometric analysis of electron micrographs and analysis of actin and myosin by gel electrophoresis. In summary, the myocardium undergoes marked, parallel changes in structure, function and biochemical composition in response to the removal and restoration of load.

Oncocytic cardiomyopathy of infancy with Wolff-Parkinson-White syndrome and ectopic foci causing tachydysrhythmias in children

Two female infants, ages 6 months and 13 months, were first seen in the newborn period with supraventricular tachycardia associated with Wolff-Parkinson-White syndrome. One infant had echocardiographic and angiographic evidence of diffuse cardiomyopathy and died suddenly at home. The other infant was seen initially at 13 months of age with refractory ventricular tachycardia and died following surgical resection of arrhythmogenic foci on the left and right ventricles. Autopsy showed diffuse patchy oncocytic cardiomyopathy in both instances. Serial histologic sections of the cardiac conduction system showed oncocytic involvement of the atrioventricular (AV) node, His bundle, and bundle branches. Both infants had interruption of the anulus fibrosus by oncocytic cells at several sites, resulting in multiple accessory AV and nodoventricular connections. Additionally, patient No. 1 had an accessory AV connection by oncocytic cells in the fatty fibrous tissue of the left AV sulcus. To our knowledge, this is the first report of multiple accessory AV connections of oncocytic cells seen during histologic study. In addition, both infants had oncocytic involvement of the exocrine and endocrine glands. This report discusses the clinicopathologic correlations in these two patients, the literature on oncocytic cardiomyopathy, and the types of dysrhythmias found in these patients and their management.

Complete reversibility of cat right ventricular chronic progressive pressure overload.

Chronic, progressive pressure overload of the cat right ventricle produces persistent, ongoing abnormalities of contractile, energetic, and biochemical function in vitro at a time when in vivo pump function is still normal. The present study tested the reversibility of the in vitro changes in this clinically relevant hypertrophy model. Fourteen sham-operated and 14 reversal cats were studied. After banding the animals as 1-kg kittens, right ventricular pressures were normal. Before band removal (25.2 ± 0.5 weeks later for the control group and 25.5 ± 0.3 weeks later for the hypertrophy reversal group), systolic right ventricular pressures were 24 ± 1 mm Hg for controls and 71 ± 5 mm Hg for the hypertrophy reversal group (P < 0.05). At study, 19.5 ± 1.1 weeks after a second sham operation for controls or 18.7 ± 0.7 weeks after band removal for the hypertrophy reversal group, these pressures were 24 ± 1 mm Hg for controls and 23 ± 1 mm Hg for the hypertrophy reversal group (P = NS); cardiac output was 0.18 ± 0.01 liters/kg per min for controls and 0.19 ± 0.01 liters/kg per min for the hypertrophy reversal group (P = NS). The ratio of right ventricle to body weight was normal in both groups, as was the right ventricular papillary muscle myocyte cross-sectional area and the myocardial collagen concentration. A right ventricular papillary muscle from each cat was studied at 29°C in a polarographic myograph. Preloaded shortening velocity was 0.79 ± 0.04 muscle lengths/sec for controls and 0.86 ± 0.03 muscle lengths/sec for the hypertrophy reversal group (P = NS); extent of shortening was 0.15 ± 0.01 muscle lengths for controls and 0.16 ± 0.01 muscle lengths for the hypertrophy reversal group (P = NS). At optimum isometric length, active tension was 59.7 ± 3 1 mN/mm for controls and 57.0 ±1.9 mN/mm22 for the hypertrophy reversal group (P = NS); resting tension was 15.6 ±1.2 mN/mm2 for controls and 13.6 ±1.6 mN/mm2 for the hypertrophy reversal group (P = NS). Active and resting oxygen consumption levels did not differ in the two groups. This study demonstrates that—in the compensated stage of chronic, progressive pressure overload of the cat right ventricle—the contractile, energetic, and biochemical abnormalities of the hypertrophied myocardium are fully reversible.

An ultrastructural morphometric study of the papillary muscle of the right ventricle of the cat

SummaryThe papillary muscle of the cat hearts right ventricle has not been studied previously with quantitative ultrastructural techniques despite its wide use for functional studies. This tissue was perfusion-fixed, processed for electron microscopy, and morphometric techniques were used to assess the ultrastructural characteristics of the papillary muscle as well as the working myocardial cells. The results of this study were that 73.5% of the papillary muscle was composed of muscle cells, 9.7% of blood vessels, and the remainder of interstitial connective tissue. In the muscle cell the volume fraction of mitochondria was 17.3%, that of myofibrils was 49.8%, and that of the nucleus was 2.0%. The mitochondria to myofibrils ratio was 0.36 and the surface to volume ratio was 0.309. In a quantitative ultrastructural comparison of perfusion and immersion fixed tissue it was found that significant differences in the volume density of the blood vessel lumen existed between the two groups. In addition, there were significant differences in the volume fraction of mitochondria and nucleus between perfusion-fixed and immersion-fixed muscle cells. A concurrent significant decrease between the two groups was also found for the ratio of mitochondria to myofibrils. The perfusion-fixed tissue can be considered to provide only normal baseline data for the papillary muscle of the right ventricle. These data are important as they can be used in future structure-function studies on normal and pathological heart tissue.

Localization of proliferating cell nuclear antigen in the developing and mature rat heart cell

The cardiac muscle cell ceases to divide shortly after birth; this cessation is followed by a limited period when DNA synthesis and karyokinesis occur without cytokinesis. The regulation of this process is not known. The purpose of this study is to explore the possible events that could lead to the cessation of cardiac muscle cell division. One protein requisite for DNA synthesis is proliferating cell nuclear antigen (PCNA). This protein is the auxiliary protein of DNA polymerase δ.

Probit analysis of the atrioventricular (AV) junctional tissues of the ferret heart

SummaryProbit frequency analysis, a graphic method for determining whether a population is normally distributed, skewed, or multinodal, was used to determine whether P cells are present in different regions of the AV junction in the ferret heart. This analysis indicated that at least 95% of the cells of the transitional zone, superficial AV node, deep AV node, and distal AV bundle of the ferret heart are morphologically homogeneous. In the proximal AV bundle a large cell population is found in addition to the AV bundle cells. The probit analysis was also used to characterize the shape of the cells of each region of the AV junction further. AV nodal cells are not as elongated as the atrial muscle cells and AV bundle cells. These nodal cells also do not branch as extensively as the AV bundle cells.